Mitochondrial diseases occur at a rate of 1 in 5000 live births and are often fatal by ~5 years old. These mitochondrial encephalomyopathies are usually oxidative phosphorylation (OXPHOS) disorders in which one or more components of the electron transport chain (ETC) are no longer operating efficiently to synthesize ATP. Although genetic sequencing has identified >200 of the underlying causative mutations in mitochondrial or nuclear DNA, a definitive biochemical link between the genetic defect and the neurodegenerative pathology remains to be elucidated. As a result there are no accurate disease biomarkers and infants are often initially identified as a result of their failure to thriv or by the existence of severe developmental delays. The innovative studies proposed here are expected to reveal a novel metabolic link between reduced OXPHOS and neuropathology, and may have utility to act as a diagnostic biomarker of mitochondrial disease. Previously, we have detected a new chemical modification of proteins in diabetes, S-(2-succino)cysteine (2SC), which is formed by reaction of the Krebs cycle intermediate fumarate with reactive cysteine residues in protein. Both fumarate and protein succination are increased in adipocytes cultured in high glucose and in diabetic mouse adipose tissue. We have shown that increased succination can significantly affect protein regulation by lowering enzymatic activity and interfering with the secretory structure of hormones. In diabetes, the increase in 2SC-modified proteins occurs as a result of nutrient excess, accumulation of NADH, and feedback inhibition of the Krebs cycle dehydrogenases, allowing the increase in fumarate. In a novel, lateral extension of these observations we propose that a similar ETC inhibition e.g. as a consequence of Complex I deficiency during Leigh Syndrome, would result in increased NADH, fumarate and protein succination in mitochondrial disease. In Preliminary Data, we demonstrate that increased protein succination is detectable on a range of proteins in the brainstem of a mouse model of Leigh syndrome (Ndufs4 knockout) in association with neuropathology. We hypothesize that the increased 2SC levels are also detectable in serum or urine during Leigh syndrome and may provide a more useful measure of metabolic derangement in the newborn than the current lactate measurements. In this study we will optimize the measurement of 2SC in serum and urine to determine its usefulness as a biomarker of OXPHOS deficiencies. We will also explore a therapeutic intervention which should lower NADH levels, reduce fumarate and thereby prevent increased protein succination, opening novel therapeutic avenues for the future treatment of mitochondrial diseases.